A molecular genetic approach will be used to analyze a set of interacting regulatory mechanisms in bacteriophage P22. Most P22 genes are subject to negative control by c2 repressor (product of gene c2). The P22 ant gene codes for a novel regulator of gene expression, antirepressor, which interacts with and inhibits c2 repressor. Synthesis of antirepresor is in turn under negative control by two repressors, the products of the arc and mnt genes. We have evidence that antirepressor synthesis is also regulated at a post-transcriptional level. We plan to isolate, sequence, and characterize mutations in the promoter-distal portion of the ant operon which reduce the rate of antirepressor synthesis and to determine whether these mutations affect ant gene expression by altering the structure of ant messenger RNA. In order to study the mechanism of repression of antirepressor, we will isolate and characterize mutations which decrease the affinity of the Oant operator for the Mnt and Arc repressors. We will construct plasmids which overproduce the Mnt and Arc proteins to facilitate the purification of both repressors, whose interaction with wild-type and mutant Oant operators will be studied biochemically in collaboration with Dr. R.T. Sauer. A sensitive and quantitative assay for the Mnt repressor will be developed and used to study the regulation of mnt gene expression in vivo. Finally, a genetic approach will be used to determine why overproduction of antirepressor interferes withe expression of other phage genes which are essential for lytic phage development. The long-term goal of this research is an understanding of the mechanisms of action of a complex set of interacting regulators of gene expression. The understanding of gene regulation is central to our understanding of normal development in higher organisms and of the diseases which result when these regulatory processes fail.